GUIDE-Seq for Genome-wide Off-target Analysis

We performed GUIDE-Seq as described previously.^{21 (link)} Briefly, HEK293T cells were electroporated in a 24-well plate with 500 ng of Cas9, 500 ng of sgRNA, 10 ng of mCherry plasmids, and 7.5 pmol of annealed GUIDE-Seq oligonucleotide using the Neon nucleofection system (Thermo Fisher Scientific). After 72 hours post-nucleofection, genomic DNA was extracted with a DNeasy Blood and Tissue kit (Qiagen 69504) according to the manufacturer’s protocol. DNA libraries were prepared using custom oligonucleotides described in Tsai, et al.^{21 (link)} Library preparations were done with original adaptors with each library barcoded for pooled sequencing. The barcoded, purified libraries were sequenced on a MiniSeq platform in a paired-end (150/150) run.
Raw sequencer output (BCL) was demultiplexed and aligned to hg38 using GS-Preprocess (github.com/umasstr/GS-Preprocess).^{43 (link)} This software also constructed a reference of UMIs unique to each read and merged technical replicate BAM files. Off-target analysis of this input was performed using the GUIDEseq Bioconductor package.^{44 (link)} Only sites that harbored a sequence with ≤10 mismatches relative to the gRNA were considered potential off-target sites. GUIDE-Seq read count data is indicated in Supplementary Figures 2–3.